Selecting a multiplex PCR panel for accurate molecular diagnosis of intestinal protists: a comparative study of Allplex^® (Seegene^®), G-DiaParaTrio (Diagenode^®), and RIDA^®GENE (R-Biopharm^®) assays and microscopic examination

Abstract

Commercial multiplex PCR assay panels were developed to overcome the limitations of microscopic examination for parasitological diagnosis on stool samples. However, given the increased supply of this diagnostic approach, these assays must be evaluated to position them in a diagnostic algorithm. Analytical performances of the multiplex PCR assay G-DiaParaTrio, Allplex® GI parasite and RIDA®GENE parasitic stool panel for detecting Blastocystis sp., Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., Dientamoeba fragilis, and Cyclospora cayetanensis, were assessed through a retrospective comparative study on 184 stool samples initially sent for parasitological investigation. The composite reference method for parasitological diagnosis was microscopic observation and Entamoeba histolytica-specific adhesion detection when necessary. Multiplex PCR assays were performed on extracted DNA from each stool, following the manufacturer’s recommendations. Discrepant results with the composite reference method were investigated with species-specific PCR to approach a final parasitological diagnosis. Overall sensitivity/specificity for the multiplex PCR assays was 93.2%/100% for G-DiaParaTrio, 96.5%/98.3% for Allplex® GI parasite and 89.6%/98.3% for RIDA®GENE, whereas the composite reference method presented an overall sensitivity/specificity of 59.6%/99.8%. These results confirmed the added diagnostic value of the multiplex PCR approach for gastrointestinal protists. Nevertheless, the PCR procedure and the analytical performance for each protist of interest, variable depending on the multiplex PCR assay, must be considered when implementing a PCR-based diagnostic approach.

Development, Optimisation and Validation of a Novel Multiplex Real-Time PCR Method for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis

“…This figure is slightly lower than those (ca. 1) reported using commercially available multiplex qPCR assays, including the Allplex GI-Parasite (Seegene, Soul, South Korea) [33,34], the EasyScreen Enteric Parasite (Genetic Signatures, Sydney, Australia) [27], the G-DiaParaTrio (Diagenode Diagnostics, Liège, Belgium) [34] and the NanoCHIP (Savyon Diagnostics, Ashdod, Israel) [30] assays. The slightly lower sensitivity of our multiplex qPCR assay is likely due to the single-copy nature of the targeted gene (cowp1) compared to the multiple-copy nature of ssu rRNA and ITS.…”

Occurrence of Cryptosporidium in human stool samples in Qatar

“…These PCR assays offer several advantages over traditional methods. Firstly, they provide increased sensitivity and speci city, particularly in populations with low rate of parasitic infections, Allplex™ GI-Parasite Assay showed high sensitivity 96.5% and speci city 98.3% (41). Secondly, the possibility of multiplex PCR assays allows the detection of various organisms and species using multiplex gene targets.…”

Evaluation of Commercial Concentration Methods for Microscopic Diagnosis of Protozoa and Helminths in Human Stool Samples in a Non-Endemic Area

“…Nowadays, the use of microscopic techniques could appear outdated, given the increasing availability of molecular tools [12,13]. In fact, several multiplex PCR assays have been developed for protozoa detection with performances equal or superior to those of microscopy [14][15][16], and they are able to detect up to six different targets [17,18]. However regarding helminth detection, commercial multiplex PCR assays have not yet surpassed microscopy in terms of performance [19,20] due to several reasons: (i) the number of parasite eggs spread in feces is usually lower than that in the case of protozoa; (ii) DNA extraction for helminth PCR examination requires adapted mechanical pretreatment to ensure wall disruption without degrading DNA [21]; (iii) the spectrum of human pathogenic helminths is much wider than that of pathogenic protozoa, which makes it difficult to design a multiplex PCR assay covering all infections.…”